Abstract

Cardiomyocyte β₂-adrenergic receptors (β-ARs) provide a source of inotropic support and influence the evolution of heart failure. Recent studies identify distinct mechanisms for β₂-AR actions in neonatal and adult rat cardiomyocytes. This study examines whether ontogenic changes in cardiac β₂-AR actions can be attributed to altered Gi expression or changes in the spatial organization of the β₂-AR complex in membrane subdomains (caveolae). We show that β₂-ARs increase cAMP, calcium, and contractile amplitude in a pertussis toxin (PTX)-insensitive manner in neonatal cardiomyocytes. This is not caused by lack of Gi; Gαi expression is higher in neonatal cardiomyocytes than in those of adult rats. β₂-ARs provide inotropic support without detectably increasing cAMP, in adult cardiomyocytes. This cannot be attributed to dual coupling of β₂-ARs to Gs and Gi, because β₂-ARs do not promote cAMP accumulation in PTX-pretreated adult cardiomyocytes. Spatial segregation of β₂-ARs, Gαs/Gαi, and adenylyl cyclase to distinct membrane subdomains also is not a factor, because all of these proteins copurify in caveolin-3-enriched vesicles isolated from adult cardiomyocytes. However, these studies demonstrate that enzyme-based protocols routinely used to isolate ventricular cardiomyocytes lead to proteolysis of β-ARs. The functional consequences of this limited β-AR proteolysis is uncertain, because truncated β₁-ARs promote cAMP accumulation and truncated β₂-ARs provide inotropic support in adult cardiomyocytes. Collectively, these studies indicate that components of the β₂-AR signaling complex compartmentalize to restricted membrane subdomains in adult rat cardiomyocytes. Neither compartmentalization nor changes in Gi expression fully explain the ontogenic changes in β₂-AR responsiveness in the rat ventricle.